EICOSANOID BIOSYNTHESIS DEFICIENCY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 基本假设：确诊的患者存在胞浆PLA2酶突变。 第二假设：确诊患者存在胞浆磷脂酶A2调节机制缺陷。 我们已经发现一名患者的多种前列腺素(环氧合酶途径的产物)和12-羟基二十碳四烯酸(12-HETE)(脂氧合酶途径的产物)水平显著降低，提示PLA2酶活性缺乏。我们试图进一步阐明这些生化缺陷的分子机制。我们将首先对常见的环氧合酶和脂氧合酶途径的血清和尿液代谢物进行定量，此外还将进行血小板聚集和磷脂特性的功能分析，以确认该个体的分子缺陷。在确认酶缺乏后，我们将通过蛋白质的蛋白质印迹分析来评估该酶是否完全不存在于细胞中或以改变的形式存在。然后，我们将在基因水平上进行描述。 PLA2在人体中的调节涉及一个复杂的过程，包括钙释放调节的激活、磷酸化、转位以及多种其他调节因子，如细胞因子、生长因子和癌基因。由于人类胞液和分泌的PLA2基因已经被绘制出来，并且功能域被很好地描述，这些酶的基因分型将有助于准确地定位我们患者功能缺陷的机制，并界定PLA2酶本身的缺陷或调节机制的缺陷。在分子和遗传水平上确定这名患者的缺陷将大大有助于了解PLA2酶在人类中的作用及其抑制的临床后果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Primary Hypothesis: The identified patient has a mutation of the cytosolic PLA2 enzyme. Secondary Hypothesis: The identified patient has a defect in regulatory mechanism of cytosolic PLA2. We have identified a patient with significantly low levels of multiple measured prostaglandins, products of the cyclooxygenase pathway, and 12-hydroxy-eicosatetraenoic acid (12-HETE), a product of the lipoxygenase pathway, suggesting a functional PLA2 enzymatic deficiency. We seek to further elucidate the molecular mechanism for these biochemical deficiencies. We will initially quantify serum and urinary metabolites of common cyclooxygenase and lipoxygenase pathways, in addition to functional analysis of platelet aggregation and phospholipid characterization to confirm the molecular defect in this individual. Following confirmation of the enzymatic deficiency, we will assess whether the enzyme is entirely absent from the cells or present in an altered form by performing Western blot analysis of the protein. We will then pursue description on a genetic level. PLA2 regulation in humans involves a complex process including calcium release-regulated activation, phosphorylation, translocation, and multiple other modulatory factors such as cytokines, growth factors, and oncogenes. Because human cytosolic and secretory PLA2 genes have been mapped and functional domains well described, genotyping of these enzymes will assist in pinpointing the mechanism of functional deficiency in our patient and delineating between a defect in the PLA2 enzyme itself or in a regulating mechanism. Defining the deficiency in this patient on molecular and genetic levels will contribute significantly to the knowledge of the role of PLA2 enzymes in humans and clinical consequences of their inhibition.